The main psychoactive component of marijuana is known as delta9-tetrahydrocannabinol (THC). In addition, endogenous substances are synthesized in the brain that can activate cannabinoid receptors, and these substances are referred to as endocannabinoids (eCB). All drugs, both natural and synthetic, that act at receptors for this substance are known collectively as cannabinoids (CBs). Cannabinoid drugs obtained by the smoking or ingestion of marijuana are used because they are reinforcing or rewarding to humans through interaction with the brain's reward circuitry. One of the objectives of these studies is to gain knowledge about the underlying mechanisms through which cannabinoids alter brain cell function, and ultimately the mechanisms that produce the pleasurable effects of these drugs that sustain their use. The primary focus of this laboratory is to examine the mechanisms through which abused drugs alter the electrical activity of neurons and the ways in which these neurons communicate with each other via synaptic connections. Therefore, one of our goals is to identify specific ion channels whose activity is modified by abused drugs such as marijuana, nicotine, heroin, and cocaine. To achieve these goals we utilize rat brain slices acutely obtained from discrete brain areas involved in processing information regarding pleasurable and unpleasant environmental stimuli. We utilize whole-cell electrophysiological recordings, and cellular anatomical techniques to reconstruct the neurons from which we record. In these ongoing studies we are examining the mechanisms through which these drugs affect neurons and their connections in the ventral tegmental area (VTA). This brain area and its connections are strongly implicated in the reinforcing and rewarding actions of all abused drugs, as well as in mediating the rewarding effects of natural environmental stimuli, such as food, water, etc. The VTA is also involved in processing information regarding the physiological stress responses, mood and affect, and mental alertness. Because of its central role in these processes, the VTA is a brain area that contributes to disorders such as addiction, psychiatric stress disorders, clinical depression, and psychiatric anxiety disorders. Our recent work in the VTA is designed understand the mechanisms through which eCBs modulate VTA circuitry during psychostimulant exposure. Many studies show that antagonism of cannabinoid CB1Rs can block self-administration of several abused drugs. Furthermore, the increase in DA concentration that is observed in the nucleus accumbens (NAc) following systemic nicotine, cocaine or ethanol administration is greatly reduced by systemic administration of the CB1R antagonist/partial agonist SR141716A2. This drug also decreases DA release in the NAc in response to the presentation of reward-associated cues, suggesting that eCBs are important for DA increases in the mesolimbic system by unconditioned and conditioned stimuli associated with drug abuse. There is also evidence that infusion of CB1R antagonists directly into the VTA can reduce the rewarding effect of peripheral nicotine, and reduce the release of DA in the VTA following systemic cocaine administration in rats. Data from our laboratory and others also show that eCBs are released from dopamine neurons in the VTA. Collectively, these data suggest that these eCBs act within the VTA on CB1Rs to increase the release of DA in the NAc during the intake of abused drugs, or during exposure to drug-paired stimuli. Although CB1Rs are located on both GABAergic and glutamatergic axon terminals within the VTA, we hypothesize that it is the inhibition of GABA release and disinhibition of DA neurons by THC that is responsible for its rewarding effects. We further hypothesize that the increase in DA neuron activity by abused drugs causes release of the eCB, 2-aracchidonoylglycerol (2-AG), which then augments DA neuron excitation through inhibition of GABA release, in an eCB-driven positive feedback loop. For these reasons we are conducting experiments to determine whether abused drugs trigger eCB release in the VTA, and what mechanisms might underlie this action. The most sensitive physiological measure of eCB function that we have found in the VTA are synaptic GABAB-receptor-mediated IPSCs, activated by electrical stimulation. These responses are strongly inhibited by presynaptic CB1Rs, and are tonically inhibited by the eCB, 2-AG, since its amplitude increases in the presence of CB1R antagonists, or when 2-AG synthesis is inhibited by inclusion of a diacylglycerol lipase-alpha; (DGL-alpha) inhibitor, THL, in the whole-cell pipettes. Additionally, phasic, activity-dependent 2-AG release can be observed when DA neurons are transiently depolarized to fire action potentials, and this is also mediated by an increase in 2-AG release. Our recently acquired data show that GABA-B IPSCs in VTA DA neurons are inhibited by cocaine (10 microM), and this is reduced by the CB1R antagonist AM251 (2 microM). The effect of cocaine is partly mediated by 2-AG released from the recorded neuron, and from surrounding cells, since THL blocks the effect more strongly when applied extracellularly, versus intracellularly. These data suggest that cocaine stimulates the release of 2-AG in the VTA to reduce GABA release onto DA neurons. We hypothesize that the reduction of inhibition by cocaine increases the excitability of midbrain DA neurons to augment release of DA in projection areas like the NAc, as described in in vivo studies. Because this cocaine action may be important for its rewarding and addicting properties, we are performing additional experiments to determine the mechanism(s) by which cocaine releases 2-AG in the VTA. Thus, in the next fiscal year we will address this issue more directly by examining the effects of cocaine on Ca2+ dynamics in VTA DA neurons in brain slices, using confocal imaging of fluorescence emitted by genetically-encoded calcium indicator (GECI) proteins. We use the floxed GECIs GCaMP5 (AAV2/1.hSynap.Flex.GCaMP5G) or GCaMP6 (AAV1.CAG.Flex.GCaMP6f)61, injected bilaterally into the VTA of THCre expressing rat lines. Our preliminary data in THCre mice indicate excellent expression of these proteins, permitting measurement of fluorescence triggered by baseline Ca2+ oscillations, or during cocaine application. In most VTA DA neurons (80%), acute cocaine (10 M) reduces Ca2+ fluorescence, and this is blocked by D2 antagonist. However, in approximately 20% of the imaged VTA DA neurons, we observe a significant increase in Ca2+ signal during cocaine application, consistent with an increase in VTA DA neuron single unit activity with cocaine in vivo. We hypothesize that the cocaine-induced increase in the activity of these DA neurons causes release of 2-AG through a Ca2+-dependent mechanism, and that these cells will show greater inhibition of synaptic GABA by 2-AG. Thus, the DA neurons showing the largest cocaine-induced increase in Ca2+ signal will also show the largest increase in GABA-B IPSCs upon CB1R antagonism. To test this we will identify VTA DA neurons showing increased or decreased Ca2+-induced fluorescence following cocaine application in brain slices from THCre rats that have received intra-VTA injections of the AAV-GCaMP6 construct. We will then perform patch clamp recordings from these 2 groups of cells, record GABAB IPSCs, and evaluate the magnitude of the cocaine-induced inhibition of the synaptic response, and determine the dependence of the cocaine inhibition with a neutral CB1R antagonist (PIMSR1, 1 M). These experiments should establish a link between cocaine use and eCB release in the VTA, and identify a novel mechanism in which cocaine dependency may be disrupted.